Phytochemical analysis, in-vitro and in-silico study of antiproliferative activity of ethyl acetate fraction of Launaea cornuta (Hochst. ex Oliv. & Hiern) C. Jeffrey against human cervical ca

Inyani John Lino Lagu1*, Dorothy Wavinya Nyamai2 and
Sospeter Ngoci Njeru3*

1Department of Molecular Biology and Biotechnology, Pan African University Institute for Basic Sciences,
Technology and Innovation, Nairobi, Kenya, 2Department of Biochemistry, School of Biomedical
Sciences, College of Health Sciences, Jomo Kenyatta University of Agriculture and Technology, Nairobi,
Kenya, 3Centre for Traditional Medicine and Drug Research (CTMDR), Kenya Medical Institute (KEMRI),
Nairobi, Kenya

Introduction: Cervical cancer is one of the leading causes of death among
women globally due to the limitation of current treatment methods and their
associated adverse side effects. Launaea cornuta is used as traditional medicine
for the treatment of a variety of diseases including cancer. However, there is no
scientific validation on the antiproliferative activity of L. cornuta against
cervical cancer.

Objective: This study aimed to evaluate the selective antiproliferative, cytotoxic
and antimigratory effects. L. cornuta and to explore its therapeutical mechanisms
in human cervical cancer cell lines (HeLa-229) through a network
analysis approach.

Materials and methods: The cytotoxic effect of L. cornuta ethyl acetate fraction
on the proliferation of cervical cancer cells was evaluated by 3-(4, 5-
dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) bioassay and
the antimigratory effect was assessed by wound healing assays. Compounds
were analysed using the qualitative colour method and gas chromatographymass
spectroscopy (GC-MS). Subsequently, bioinformatic analyses, including the
protein-protein interaction (PPI) network analysis, Gene Ontology (GO), and
Kyoto Encyclopaedia of Genes and Genomes (KEGG) analysis, were
performed to screen for potential anticervical cancer therapeutic target genes
of L. cornuta. Molecular docking (MD) was performed to predict and understand
the molecular interactions of the ligands against cervical cancer. Reverse
transcription-quantitative polymerase chain reaction (RT-qPCR) was
performed to validate the network analysis results.

Introduction

Cervical cancer is a major global health disease that affects
women (Drokow et al., 2022). It is one of the leading causes of cancer
death among women and the fourth most common cancer
worldwide (Revathidevi et al., 2021). Human papillomavirus is
the primary aetiological driver in carcinogenesis
(Balasubramaniam et al., 2019). Not all human papillomavirus
(HPV) infections in women result in cervical cancer. High-risk
HPV genotypes induce a normal cell to transform into a
precancerous lesion, which subsequently becomes an invasive
lesion (Kasi et al., 2021). HPV infection causes the
overexpression of viral oncogenes, which may inhibit a number
of cellular proteins and affect biological processes such as cell
proliferation, cell cycle, and apoptosis (Martínez-Rodríguez
et al., 2021).
Globally, in 2020, a total of 604127 cases of cervical cancer were
estimated, and 341831 deaths were recorded (Olthof et al., 2024).
The incidence rate was 13.3 cases per 100,000 women, and the
mortality rate was 7.2 deaths per 100,000 women (Singh et al., 2023).
Over 58% of cervical cancer cases globally were estimated in Asia,
followed by Africa (20%), Europe (10%) and Latin America (10%)
(Singh et al., 2023). However, the incidence and mortality rates for
cervical cancer are exceptionally high in Sub-Saharan Africa at
19.59% and 24.55% annual global burden, respectively (Black and
Richmond, 2018).
Botanicals’ secondary metabolites and their derivatives have
been evaluated in oncology-related clinical research and trials
(Omara et al., 2022). The comprehensive knowledge on the
mechanism of action for some investigated plant compounds
provided the basis to develop novel, efficacious, safer botanicalderived
compounds or their semi-synthetic analogues. Such had
additional therapeutic advantages over the convectional
chemotherapeutic drugs, which are associated with high toxicity
not only to cancer cells but also to noncancerous cells (Scatchard
et al., 2012; Vordermark, 2016; Johnson et al., 2018; Paken et al.,
2023). Cervical cancer has become resistant to conventional
therapeutic drugs, including cisplatin, paclitaxel, and carboplatin
(Small et al., 2017), thus rendering them less effective. Hence, it was
imperative to speed up research on botanical plants as an alternative
source of less toxic, more tolerable and effective anticancer drugs for
cervical cancer treatment.
The rich biodiversity has contributed significantly to
traditional medicine and medicinal systems since ancient
times (Omara, 2020). Approximately 1,200 medicinal plants
are used in Kenya, 41 of which have anticancer properties,
which account for only 10% of the medicinal plants
(Onyancha et al., 2018). Botanical-based drugs are gaining
attention due to their low toxicity, availability, costeffectiveness,
and tolerability compared to conventional
drugs (Tariq et al., 2017; Ochwang’i et al., 2014; Misonge
et al., 2016). In this study, Launaea cornuta (Hochst. ex Oliv.
& Hiern) C. Jeffrey also known as “mchunga” was investigated.
L. cornuta is an erect herbaceous perennial plant belonging to
the Asteraceae family, characterised by a hollow leafy and stem,
slightly succulent, glabrous, with milky juice and grows to a
height of about 1.5 m (J et al., 2015). It is indigenous to Kenya,
Burundi, Cameroon, Uganda, Nigeria, Rwanda, Chad, Djibouti,
Eritrea, Ethiopia, Malawi, Mozambique, Somalia, Sudan,
Tanzania, Zambia, Central African Republic, Zaïre, and
Zimbabwe (Machocho et al., 2014; Omara et al., 2022). In
Kenyan communities, this plant is used as a wild vegetable

and a source of vitamin C (Musila et al., 2013). The localised
budding root concoction known as ‘Kipche’ is used to cure
throat cancer (“Koroitab mokto”), typhoid, gonorrhoea, benign
prostate hyperplasia, and breast cancer, among others (J et al.,
2015; Fatuma Some, 2014; Khan et al., 2016; Chemweno et al.,
2022). Regardless of the extensive use of L. cornuta plants in
traditional medicine, their therapeutic efficacy, and toxicity
have not been empirically validated, especially in
cervical cancer.
In this study, we systemically evaluated the anticancer activity of
the ethyl acetate fraction of L. cornuta, analysed and identified
compounds using the GC-MS approach and explored the relevant
targets and pathways involved in eliciting the anticervical effects
through a network analysis approach.

Modified Eagle Medium (EMEM) supplemented with 1%
L-glutamine (200 mM), 10% fetal bovine serum (FBS), 1.5%
sodium bicarbonate, 1% HEPES (1M), 1% penicillinstreptomycin
and 0.25 μg/mL amphotericin B. The cell culture
was conducted at 37°C under a humidified atmosphere and 5%
CO2 to achieve 75% – 80% confluence.